Method and composition for inhibiting cholesterol esterase

ABSTRACT

This invention encompasses methods for manufacturing purified, high molecular weight sulfated polysaccharide compositions that inhibit pancreatic cholesterol esterase and lower cholesterol in the blood stream.

This is a continuation application of application Ser. No. 08/816,823filed on Mar. 18, 1997 now U.S. Pat. No. 6,632,801, which is acontinuation of application Ser. No. 08/451,563, filed May 26, 1995, nowabandoned which is a divisional of application Ser. No. 08/322,782 filedon Oct. 13, 1994, now U.S. Pat. No. 5,521,303.

BACKGROUND OF THE INVENTION

This invention relates to a method for preparing a therapeutic agentthat upon ingestion decreases intestinal cholesterol absorption in manand specifically inhibits or decreases intestinal cholesterol absorptionby inhibiting the pancreatic cholesterol esterase catalyzed hydrolysisof naturally occurring and ingested cholesterol esters and by inhibitingthe cholesterol esterase facilitated uptake of free cholesterol.

The invention is based upon the discovery that pancreatic cholesterolesterase is an important contributor to overall dietary cholesterolabsorption because (1) cholesterol derived from cholesterol esters ispreferentially absorbed compared to free cholesterol; (2) cholesterolesterase enhances the absorption of free cholesterol and (3) dietarycholesterol and/or cholesterol esters induce the mRNA and level ofenzymatic activity of cholesterol esterase in the pancreas in a newlydiscovered intestinal-pancreatic cycle for the absorption ofcholesterol. U.S. Pat. Nos. 5,173,408 and 5,063,210 describe theimportance of cholesterol esterase in the dietary uptake of cholesteroland also disclose methods for inhibiting cholesterol esterase. Thus, thesurprising usefulness of inhibiting cholesterol esterase hasdemonstrated a new need for potent (Ki less than 5 μM) and safeinhibitors of cholesterol esterase.

Many physical ailments are attributed at least in part to high levels ofserum cholesterol. Atherosclerosis, for example, is a leading cause ofdeath in the United States and high serum cholesterol concentrations areassociated with increased risks of fatal atherosclerotic events. Thediscovery that the cholesterol esterase enzyme plays a role inintestinal cholesterol absorption has led to attempts to attenuateintestinal cholesterol absorption in man by inhibiting the action of thecholesterol esterase enzyme. As a result of these findings, there is nowan important need to develop human pancreatic cholesterol esteraseinhibitors, especially those that are not absorbed and are essentiallynondegradable. The pharmacology of various polysaccharides has beeninvestigated. Cook and Cammarata, 1963, Arch. Int. Pharmacodyn. 144: 1.In particular, crude sulfated amylopectin has been taught in U.S. Pat.No. 4,150,110 as an anti-ulcer agent, but its property as a cholesterolesterase inhibitor has not been recognized.

Sulfated dextran of low molecular weight has been recognized for use inthe treatment of hyperlipemia and as an orally administeredanticoagulant. British Patent No. 953,626. In Japan, low molecularweight sulfated dextran (MDS) at a dose of 1800 mg/day has been used toreduce serum cholesterol levels by activating a blood enzyme lipoproteinlipase. Goro et al, 1987, J. Clin. Biochem. Nutr. 2: 55–70. Asdemonstrated by carbon-14 labelling studies, the low molecular weight ofthis bacterial dextran, (7–8,000 Daltons), allows the sulfated dextranto be absorbed by the intestine Drugs In Japan (Ethical Drugs, 10th ed.1986). MDS was developed for this property of intestinal absorption asindicated by the claim that a faster reduction in serum lipids can beobtained by intravenous administration of this agent with clearance ofserum lipemia due to activation of plasma lipoprotein lipase. Clearlythis route of administration will not lead to effects on inhibitingcholesterol esterase in the intestine. Absorption of MDS can lead to avariety of side effects, most notably, anticoagulant effects that mustbe monitored. This preparation has not been known to inhibit cholesterolesterase and it is sulfated randomly and at various ring positions. Highmolecular weight dextran sulfate has been excluded from development byothers because of its lack of absorption and its attendant inability toactivate serum lipoprotein lipase.

More recently, it has been discovered that crude non-absorbablepolysaccharides sulfated at the three position of the glucopyranose ringare effective as inhibitors of cholesterol esterase. See U.S. patentapplication Ser. No. 08/121,369. Useful 3-sulfated polysaccharides maybe derived from the synthetic sulfation of polysaccharides from variousnatural sources including seaweeds.

Methods for preparing sulfated polysaccharides are also known in theart. For example, U.S. Pat. No. 3,624,069 describes the sulfation ofcellulose with a sulfur trioxide/lower n-dialkyl amide sulfationcomplex. U.S. Pat. No. 4,480,091 describes a process for preparingcellulose sulfate esters in a three step process. Finally, U.S. Pat. No.4,814,437 describes a method for preparing sulfated polysaccharides bysubjecting the polysaccharide to a reducing step prior to sulfation.

SUMMARY OF THE INVENTION

The present invention is directed to a method for manufacturing highmolecular weight 3-sulfated polysaccharides that are essentiallynon-absorbable and nondegradable in the alimentary tract, and whenadministered orally, they are useful in decreasing human serumcholesterol and LDL levels by inhibiting human pancreatic cholesterolesterase, now recognized as a key enzyme involved in mediatingcholesterol absorption. Thus, following the methods of this invention asulfated polysaccharide compound is prepared wherein greater than 95% ofthe compound has a molecular weight greater than 75,000 Daltons, thesulfate to monomer ratio is between 1.0 and 3.0, and less than 0.5% byweight of the material is free sulfate. The very high molecular weightsulfated polysaccharides manufactured by methods of this invention canbe administered to humans in tablet form, incorporated in a foodstuff,or by any other method that inhibits cholesterol absorption in thealimentary tract.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a ¹³C-NMR spectrum of a very high molecular weight sulfatedpolysaccharide of this invention;

FIG. 2 shows possible structures of sulfated cellulose of thisinvention;

FIG. 3 is a FITR spectrum of a very high molecular weight sulfatedpolysaccharide of this invention; and

FIG. 4 is a plot of cholesterol uptake in Caco-2 cells over time.

DESCRIPTION OF THE PREFERRED EMBODIMENT

In accordance with the present invention, we have made certaindiscoveries concerning approaches to inhibiting cholesterol absorptionfrom the intestine to reduce the level of serum cholesterol and theincidence of atherosclerosis. Previously, a lack of understanding of therole that cholesterol esters play in the diet has precluded developmentof effective inhibitors of cholesterol esterase. Cholesterol derivedfrom cholesterol esters represents only 10 to 15% of total dietarysterol that is absorbed, Dietschy, Intestinal Lipid Absorption inPhysiology of the Gastrointestinal Tract, Vol. 2 p. 1170, Raven Press,N.Y. (1981). In addition, dietary cholesterol esters are not absorbed bythe small intestine unless they are first hydrolyzed by pancreaticcholesterol esterase. Vahouny, G. & Treadwell, C. Proc. Soc. Exp. Biol.Med. 116, 496 (1964). Because of the generally accepted thesis thatcholesterol esters contribute little to the total absorbed cholesterol,little attempt has been made to inhibit the intestinal absorption ofcholesterol esters.

It has now been found that cholesterol derived from esters ispreferentially absorbed, by more than 80%, when compared to freecholesterol. In addition, cholesterol esterase also promotes theabsorption of free cholesterol. Biochemistry, 32: 12085–89 (1993). Theseobservations demonstrate that cholesterol esterase contributessignificantly to total cholesterol absorption and there is now animportant need to develop inhibitors of human pancreatic cholesterolesterase.

The present invention is a non-obvious improvement over the prior art ofthis invention, because the very high molecular weight sulfatedpolysaccharides (defined below) are (1) vastly more potent inhibitors ofcholesterol esterase than heparin and other low molecular weightpolysaccharides which, to a small extent, inhibit the enzyme, (2)non-absorbable from the intestine, (3) inexpensive, (4) morecontinuously in contact with the intestinal enzyme by virtue of (1) and(2); and (5) essentially non-toxic.

Dietary intake of cholesterol is independently linked to coronary heartdisease and hence intestinal cholesterol absorption is an important partof the lipid homeostatic process. The rate limiting step for intestinalcholesterol absorption is mediated by the cholesterol transport functionof cholesterol esterase. This protein is unique in humans because thereis a novel exon 11 in the gene, a unique C-terminal extension of theprotein and a unique inhibitory site in the primary structure. Kumar etal., 1 Biochemistry 31, 6077 (1992). Large 3-sulfated polysaccharidesbind to this unique sequence producing potent inhibition with IC₅₀'s inthe sub-nanomolar range for the human enzyme. One of these inhibitors,very high molecular weight cellulose sulfate prepared by the method ofthis invention, has an IC₅₀ of 20 pM towards the human target and100,000 pM towards rabbit cholesterol esterase. High molecular weightsulfated cellulose (1.5 million Da) is not absorbed from the intestine,and it inhibits cholesterol uptake into cultured human Caco-2 cells.Cellulose sulfate decreases serum cholesterol levels in the normalchow-fed rabbit, indicating inhibition of reabsorption of hepaticallysecreted cholesterol. In cholesterol fed rabbits, administration (100mg/kg) of very high molecular weight cellulose sulfate (1) decreasescholesterol absorption by 80%, (2) decreases serum cholesterol by over50% and (3) decreases hepatic cholesterol by over 30%. These dataindicate that small doses of cellulose sulfate having a molecular weightgreater than about 500,000 Daltons is an effective pharmaceutical agentto decrease serum cholesterol levels and LDL levels.

Free sulfate and low molecular weight sulfated polysaccharides areundesirable byproducts of the manufacture of very high molecular weightsulfated polysaccharides. In fact, the presence of toxic, low molecularweight sulfated polysaccharides or inorganic sulfate in high molecularweight sulfated polysaccharide compositions obviated their use as aningestible or injectable drug for any purpose. Therefore, the very highmolecular weight sulfated polysaccharide of this invention must includeless than 0.5 wt % free sulfate and moreover, it must contain less than5% by weight of sulfated material having a molecular weight less than75,000 Daltons.

We have found a method to recover pure, very high molecular weightsulfated polysaccharides that eliminates the toxic, low molecular weightpolysaccharide and free sulfate by products. This method produces a newvery high molecular weight sulfated polysaccharide composition of matterwhich is an extremely useful inhibitor of the cholesterol esterasemediated absorption of cholesterol.

The very high molecular weight sulfated polysaccharides of thisinvention are characterized as follows:

Property Appearance Off-White Powder Sodium content 11.0–15.0 wt %Carbon content 14.0–17.0 wt % Hydrogen content 2–3.5 wt % Nitrogencontent <0.5 wt % Sulfur content 16.0–19.0 wt % Degree of sulfation 2 ±1.0 % Free Sulfate <0.50% Specific Activity <2 × 10⁻⁴ mg/mlViscosity >4000 centipoise pH on dissolution 6–9 Wt. % with MolecularWt. >75,000 >95% Average Molecular Wt. >500,000 Daltons

Very high molecular weight sulfated polysaccharides of this inventionare made by the following steps: (1) prepare an anhydrous DMF suspensionof a high molecular weight polysaccharide or cellulose from a sourcesuch as cotton linters; (2) mix the anhydrous DMF suspension of highmolecular weight polysaccharides or cellulose with a sulfur source suchas a sulfur trioxide/DMF complex; (3) neutralize the acidic mixtureafter the sulfation reaction is essentially complete to give a crudesulfated polysaccharide mixture including crude sulfated polysaccharidesand aqueous reactants; (4) separate crude, very high molecular weightsulfated polysaccharides from the aqueous crude sulfated polysaccharidemixture; (5) wash the separated crude very high molecular weightsulfated polysaccharides; and (6) dry the resulting crude intermediateproduct.

The dried crude intermediate product is then purified to excludeessentially all impurities such as free sulfates and sulfatedpolysaccharides having a molecular weight less than 75,000 Daltons.Purification is preferably accomplished by dissolving the dried crudeintermediate product in water to form an aqueous crude solutioncontaining very high molecular weight sulfated polysaccharides andimpurities including free sulfate and low molecular weight sulfatedproducts having molecular weights less than 75,000 Daltons. The crudeaqueous solution is subjected to a first filtration step to produce avery high molecular weight sulfated polysaccharide containing filtrateessentially free of unreacted polysaccharides and/or fines. Preferablythe first filtration step consists of at least two successive filtrationsteps; the first across a 5 micron filter and the next across a smallerfilter and so forth until the final filtration step which preferablyuses a 1 micron filter.

The filtrate produced in the first filtration step is then diafilteredin a second filtration step with a 500,000 Dalton molecular weightcut-off membrane against deionized water to produce a purified very highmolecular weight sulfated polysaccharide product. The diafiltration stepeliminates free sulfates, bicarbonate, and essentially eliminates lowmolecular weight sulfated polysaccharides having molecular weights lessthan 75,000 Daltons that remain in the filtrate from the firstfiltration step. The aqueous purified product is preferably dried beforeit is used. Any drying process known in the art, such as spray drying,drum drying, fluid bed granulation, or lyophilization, that is capableof producing powder from an aqueous solution containing dissolved solidsmay be used.

In accordance with the present invention, we have made certaindiscoveries concerning structural features of very high molecular weightsulfated polysaccharide human pancreatic cholesterol esterase inhibitors(molecular weight greater than 75,000 Daltons) prepared fromnon-mammalian and non-bacterial polysaccharides. These includediscoveries as to the synthesis and characteristics of sulfatedpolysaccharides that render highly specific derivatives withsubnanomolar inhibitory constants toward human cholesterol esterase,which, along with their large size, makes them essentially nonabsorbableand non-degradable. For example, the very high molecular weight sulfatedpolysaccharides of this invention do not activate the plasma enzymelipoprotein lipase after oral use. Thus, these sulfated polysaccharidesact to reduce the cholesterol esterase facilitated absorption ofcholesterol by multiple mechanisms, for example by (1) inhibitingenzymatic cleavage of cholesterol esters, (2) displacing enzyme from itsbinding site on the intestinal cell, and (3) inhibiting transport offree cholesterol into the small intestinal cell. In addition, theseagents, unlike tetrahydrolipostatin, do not cause steatorrhea ineffective doses given to animals.

While a number of structural features can modulate the degree ofinhibition, the presence of a 3-sulfate markedly enhances inhibition.Furthermore, not all sulfated polysaccharides inhibit cholesterolesterase. Chondroitin sulfate, for example, is not inhibitory in itsnative state. The repeating unit in this polysaccharide consists of twosubstituted glucopyranose-like rings linked through the hydroxyl groupat the 3-position of one to the hydroxyl group at the 1-position of theother. Therefore, the dimeric repeat unit has only one unsubstitutedhydroxyl group at the 3-position. When this polysaccharide is sulfated,it becomes a potent inhibitor of human cholesterol esterase, indicatingthat the presence of a 3-sulfate on the glucopyranose ring is bothnecessary and sufficient for producing inhibitory activity. On the otherhand, the presence of a 2-sulfate decreases inhibition while a 6-sulfateis unnecessary.

The efficacy of sulfated polysaccharides for decreasing cholesterolabsorption is increased by reducing the absorption of the sulfatedpolysaccharide from the intestine and thus prolonging its contact withthe enzyme, among other things. Very high molecular weight sulfatedpolysaccharides are poorly absorbed and, therefore, are necessary andsufficient to inhibit the absorption of cholesterol. The increasedmolecular weight also increases the inhibitory activity of thepolysaccharides and sulfation increases the solubility and access toenzyme to produce greater inhibition. For example, low molecular weightdextran sulfate (MW=5000 Daltons) exhibited an IC₅₀ of 20 nM while theIC₅₀ of high molecular weight sulfated polysaccharides (MW=500,000Daltons) was 0.02 nM. Accordingly, the present invention includes veryhigh molecular weight sulfated polysaccharide compounds of the formula:

The chemical formula for a monomeric unit is C₆H₈Na₂O₁₁S₂, wherein n is1400 or greater and wherein R is —SO₃Na.

Cellulose sulfate is preferably used in preparing a very high molecularweight sulfated polysaccharide of this invention which is manufacturedin three basic steps: (1) sulfation of chemically pure cellulose usingsulfur trioxide in dimethyl formamide; (2) filtration to remove waterinsoluble contaminants and diafiltration against 500,000 Daltonmolecular weight or greater cut-off membranes to remove potentiallytoxic small molecular weight contaminants; and (3) an optionalformulation step to produce a tablet, capsule, liquid or foodstuffcomprising a very high molecular weight sulfated polysaccharide forhuman consumption.

The very high molecular weight sulfated polysaccharide of this inventionmay be taken in doses ranging from about 10 mg to about 5,000 mg andhigher immediately before, with, or after meals, three times per day.The very high molecular weight sulfated polysaccharide functions byinhibiting the cholesterol esterase mediated absorption of cholesterolresulting in a lowering of its concentration in human blood serum.

A preferred very high molecular weight sulfated polysaccharide of thisinvention is cellulose sulfate consisting of chemically pure cottoncellulose linters which have been sulfated in a preferred ratio of abouttwo moles of sulfate per mole of monomer. Cotton linter is a preferredsource of cellulose since it is the most chemically pure form ofcommercial cellulose yet discovered. Cotton linter consists of glucoseunits polymerized to a total of about 14.000 monomer units with amolecular weight of about 2.4 million.

In essence, our discovery leads to a practical method for convertingnaturally occurring very high molecular weight polysaccharides andpreferably cellulose polymers, often regarded as waste, into a highlypotent, cheap, non-absorbed (they do not activate plasma lipoproteinlipase after oral administration), non-toxic, and nondegradableinhibitors of cholesterol that can be administered as soluble agents insmall and well-tolerated quantities. Those skilled in the art willrecognize that methods to disperse and/or enhance or prolong thepresence in the intestine of inhibitors to increase their contact withcholesterol esterase will further decrease the absorption ofcholesterol.

The very high molecular weight sulfated polysaccharide inhibitormanufactured by the methods of this invention can also be administeredin combination with inhibitors of ACAT, acyl CoA: cholesterolacyltransferase. These compounds can lower cholesterol especially inanimals (Largis et al. 1989), but they possess a number of toxic sideeffects since they are absorbed and are not inert. Side effects can belowered by reducing their dosage while maintaining efficacy incombination with inhibitors of cholesterol esterase that are notabsorbed. A person skilled in the art will also recognize that variousACAT inhibitors, such as, for example, that described in Heider et al.,J. Lipid Res. 24: 1127 (1983), can be combined with the very highmolecular weight sulfated polysaccharides of the present invention toreduce serum levels of cholesterol.

In addition, the very high molecular weight sulfated polysaccharideinhibitors of cholesterol esterase can be administered in combinationwith cholesterol synthesis blockers. Humans treated with cholesterolsynthesis blockers experience various toxic side effects, which can bereduced by decreasing the dose administered to the patient. Therefore,administering the sulfated polysaccharide of the present invention incombination with drugs that are absorbed by the intestine and block theendogenous synthesis of cholesterol allows for decreased dosages ofcholesterol synthesis blockers to obtain the same end result. Thetoxicity associated with cholesterol synthesis blockers can beeffectively reduced while still reducing serum cholesterol levels.

Persons having skill in the art will recognize various cholesterolsynthesis blockers, such as, for example, lovastatin, which can becombined with the sulfated polysaccharides of the present invention toreduce serum levels of cholesterol.

The very high molecular weight sulfated polysaccharide inhibitors ofcholesterol esterase manufactured by the methods of this invention canbe administered in various pharmaceutical dosage forms such as tablets,capsules, liquids and powders, alone or in the presence of one or morepharmaceutical excipient such as surfactants, flavoring agents, coloringagents, starch, sugars and the like excipients. The very high molecularweight sulfated polysaccharides of this invention can also beincorporated into food products such as biscuits and cookies. Inessence, the very high molecular weight sulfated polysaccharides of thisinvention can be used as a dietary supplement to reduce cholesterolabsorption, especially from foods rich in cholesterol and/or cholesterolesters where an unexpectedly large benefit would be obtained. Thoseskilled in the food and pharmaceutical arts will recognize a widevariety of formulations and vehicles for administering sulfatedpolysaccharides.

Preferably, very high molecular weight sulfated polysaccharides areadministered to humans at or about (within about a half hour of) thetime of food intake and especially with foods that are rich incholesterol esters and/or free cholesterol. In addition, these highmolecular weight sulfated polysaccharides inhibit cholesterol introducedinto the intestine from bile.

The invention is illustrated further by the following examples which arenot to be construed as limiting the invention in scope or spirit to thespecific procedures described in them.

EXAMPLE 1

This example details a method for manufacturing a very high molecularweight sulfated polysaccharide of this invention that is useful ininhibiting cholesterol absorption.

A purified very high molecular weight sulfated polysaccharide isprepared by sulfating cellulose using sulfur trioxide dimethylformamide(DMF/SO₃) complex in anhydrous dimethylformamide (DMF) solvent accordingto the following method.

A. Dried cotton linters (8.75 kg) were shredded using a commercial papershredder and soaked in 208 liters of dry DMF under a blanket ofnitrogen. The mixture was cooled to 8–10° C.

B. After 3 hours, 33 kg of DMF/SO₃ complex were added with stirring. Thereaction temperature was maintained between 15° C. and 20° C. for 150min.

C. Solid sodium bicarbonate (51 kg) was added to the combined mixtureand allowed to mix for 10 minutes to neutralize any excess acid. Thiswas followed by 15 L of deionized water. Finally, acetone was added (95L) and the mixture stirred overnight.

D. The next day, the reaction mixture was spun in a centrifuge, and thesolid collected and resuspended in 208 L of acetone. The resuspendedmixture was spun again in the centrifuge.

E. The solid recovered from the centrifugations was dried on a dryingtable overnight.

F. The crude dried sulfated polysaccharide was dissolved in water(600–1000 L) so the solution was about 0.5–1.0 wt % solids.

G. The mixture was sequentially filtered using a 50 micron, 5 micron,and 1 micron filter. A diafiltration apparatus equipped with 500,000Dalton molecular weight cutoff membranes (Koch Membranes, pm 500 A) wasthen used to diafilter the 1 micron filtrate against deionized water toan effluent conductivity of <300 mS/cm.

H. The diafiltered solution was dried (in a spray drier or drum drier)and the resulting very high molecular weight sulfated polysaccharide ofthis invention was collected in containers of appropriate size forstorage and shipment.

The very high molecular weight sulfated polysaccharides exhibited thefollowing properties (the average values for nine manufacturing runs):

TABLE I Property Result Appearance Off White Specific Rotation ofHydrolysate 16.4° Degree of Sulfation 2.06 % Free Sulfate 0.18%Dimethylformamide 18 ppm Potency (IC₅₀) 24 ng/ml Molecular Wt. 3,800,000Daltons % Low Molecular Weight Sulfated Cellulose 0.67%

EXAMPLE 2

Nuclear magnetic resonance (NMR) spectroscopy is the standard method forstructural analysis of organic molecules. While this technique is widelyused for structure elucidation of small molecules, there are a number ofproblems which make this method of limited usefulness for largemolecules, such as the very high molecular weight sulfatedpolysaccharides of this invention.

¹³C NMR Spectra.

The ¹³C spectrum (90 MHz) of a very high molecular weight sulfatedpolysaccharide produced by the method of Example 1 is shown in FIG. 1.The eight different structural possibilities for any given saccharide ofthe very high molecular weight sulfated polysaccharides should give riseto 48 signals. (FIG. 2). However, since the observed spectrum producesonly six well-defined signals, there is much overlap, making definitiveassignments for all the carbon atoms impossible. The position andintensity of these various resonances are summarized below for thecompound of Example 1.

TABLE II ¹³C NMR CHEMICAL SHIFTS CHEMICAL SHIFT, ppm INTEGRATEDINTENSITY 100.2 10.00 78.5, 77.4 29.39 74.4, 72.5 33.00 66.0 14.85Even though some assignments are controversial, See Kamide, K. andOkajima, K. (1981) Polymer Journal p. 163–166 and Kowasaka, K. Okajima,K. and Kamide, K. (1991) Polymer Journal, p. 823–836, from studies onmodel compounds, there is agreement on the spectroscopic behavior ofcarbon 1 and carbon 6. For example, in going from β-D-glucopyranose tothe corresponding 6-sulfate derivative, signals at these two positionsshift in a characteristic way. From data on this model compound, it canbe predicted with confidence that the chemical shift at 100.2 ppmobserved in the analyzed compound is most likely due to carbon 1.Moreover, in the starting unsubstituted saccharide there is a resonancethat is shifted by 6.6 ppm in the sulfated derivative. Taken together,this indicates that the resonance in native cellulose which occurs at60.5 ppm and is shifted to 66.0 ppm on sulfation is due to carbon 6.Based on this, it may be concluded that the very high molecular weightcompound analyzed is totally sulfated at position 6 since there is noevidence of a signal at around 60.5 ppm. This is also verified by theintegrated intensity (14.85) of this signal, which corresponds to onlyone carbon atom. If the integrated intensities at 72.5 ppm, 74.4 ppm,77.4 ppm, and 78.5 ppm are summed, the total (62.4) is about four timesthat from carbon 6. This indicates that these signals are derived fromcarbons 2, 3, 4 and 5 in the various mono-, di-, tri- and unsubstitutedforms. Finally, the signal at 100.2 ppm from carbon 1, which is only ⅔the intensity of the others has a lower value because of a longerrelaxation time.

Since carbon 6 is sulfated in all the anhydro glucopyranose units, thenumber of contributing structures to the ¹³C NMR spectrum is diminished.It is also believed that the resonances from carbon 1 and sulfatedcarbon 6 are the same in all contributing structures, (See Kowansaka, K.Okajima, K. and Kamide, K. (1991) Polymer Journal, p. 823–836), reducingthe number of magnetically non-equivalent carbons from 48 to 16. Sincethere are only 4 resonances to account for in the remaining 16structures, it is still not possible to determine the relativeproportions of sulfation at carbons 2 and 3.

To summarize, it is clear that carbon 6 is, within the limits of thisanalysis, totally sulfated. Since the polysaccharide contains more thanone sulfate per monomer, the other sulfate is distributed betweencarbons 2 and 3.

EXAMPLE 3

This example details a method for isolating the human cholesterolesterase enzyme for use in testing the potency of very high molecularweight sulfated polysaccharides of this invention.

S-Sepharose Column Preparation

An S-Sepharose suspension (150 ml) was poured into a 250 ml graduatedcylinder and the gel was allowed to settle. The supernatant was thenpoured off and 100 ml of a 25 mM acetic acid solution, pH 5.1, was addedto the cylinder. The cylinder was covered with parafilm and the gelresuspended by gently inverting the graduate several times. Theresuspended S-Sepharose was poured into a column in one application andallowed to settle under gravity. When the resin settled, the bottom ofthe column was opened and the buffer was drained through the resin until1–2 cm of buffer remained over the resin bed.

S-Sepharose Chromatography

Breast milk (200 ml) stored at −20° C. and thawed to room temperaturewas transferred to a 250 ml beaker equipped with a stir bar. The pH wasadjusted to 5.1 by the dropwise addition of 1M acetic acid. The milk wascentrifuged at 15,000 rpm for 30 minutes at 4° C., and the clearsolution was carefully removed from the upper fat layer. Residual fatand insoluble material were removed by passing the solution through a0.8 micron filter.

The S-Sepharose column was filled with filtered breast milk, and thesample was applied under gravity feed. When all the sample had beenadded to the resin, the sides of the column were washed twice with 25 mlof 25 mM acetic acid, pH 5.1, followed by 400 ml of a 300 mM NaCl/25 mMacetic acid solution, pH 5.1. The absorbance at 280 nm of the effluentwas then checked using a spectrophotometer. If the absorbance wasgreater than 0.025, the resin was washed with additional 50 ml aliquotsof the 300 mM NaCl/25 mM acetic acid buffer solution until theabsorbance was less than 0.025.

Cholesterol esterase was removed from the resin at a flow rate of 60ml/hr using a 300 ml salt gradient increasing from 300 mM NaCl/25 mMacetic acid, pH 5.1, to 1.0 M NaCl/25 mM acetic acid, pH 5.1. Fractionswere collected every 2 to 4 minutes and the absorbance at 280 nm ofevery other fraction was determined as well as the enzymatic activityusing p-nitrophenyl butyrate as substrate. Momsen, W. & Brockman, H.(1977) Biochim. Biophys. Acta 486, 103–113. All fractions with ahydrolytic activity greater than 0.030 Abs/min were pooled in agraduated cylinder and the volume was doubled with 10 mM NaCl/20 mMacetic acid, pH 5.1. The sample was transferred to a dialysis tube (MWcutoff—12–14,000 Daltons) and dialyzed at 4° C. against three changes of4 L of 10 mM NaCl/25 mM acetic acid, pH 5.1.

SP-Sephadex Chromatography

SP-Sephadex C-25 (10 g) was swollen in 10 mM NaCl/25 mM acetic acid, pH5.1, and poured at 4° C. into a 2.6×40 cm glass column. The dialyzed,partially purified cholesterol esterase was pumped onto the SP-Sephadexcolumn at 60 ml/hr, and the resin was washed with 100 ml of 10 mMNaCl/25 mM acetic acid pH 5.1. The enzyme was removed with 200 mMNaCl/25 mM acetic acid, pH 5.1. Forty fractions were collected, and theabsorbance at 280 nm of every other fraction was determined as well asthe enzymatic activity using p-nitrophenyl butyrate as substrate.

Assessment of Homogeneity and Storage

Polyacrylamide gel electrophoresis (8%) was used to assess the purity ofsamples from the SP-Sephadex column by the method of Laemmli, U.K.Nature, 227: 680 (1970). To avoid overloading the gel, 0.02 opticaldensity units was removed from each fraction, using the followingformula:Volume removed (ml)=0.02/AbsProtein was visualized with the 0.2% Coomassie Brilliant Blue.Dilution and Storage of Enzyme Aliquots

Those fractions which gave a single band at 110 kDa were pooled andfrozen at −80° C. The absorbance at 280 nm of this pool was adjustedwith 200 mM NaCl/25 mM acetic acid solution, pH 5.1, to give a finalvalue of 0.070. The protein solution was then divided into 100 μlaliquots and stored frozen at −80° C. until ready for use.

EXAMPLE 4

This example describes a method for measuring the potency, (IC₅₀), ofvery high molecular weight sulfated polysaccharides.

The non-absorbable, very high molecular weight sulfated polysaccharidesof this invention are potent inhibitors of the human cholesterolesterase (CEase)—catalyzed hydrolysis of cholesterol oleate. Todetermine the IC₅₀ of inhibition, increasing amounts of sulfatedpolysaccharides are included in an enzyme assay, and the concentrationwhich produces 50% inhibition is defined as the IC₅₀.

A 1 mg/ml solution of high molecular weight sulfated polysaccharide in10 mM Tris (pH 7.5) buffer was diluted serially with 10 mM Tris (pH 7.5)to give solutions ranging in concentration from 1×10⁻¹ mg/ml to 1×10⁻⁶mg/ml. Thirty microliters of each diluted solution were added to aseries of test tubes; 30 μl of 10 mM Tris (pH 7.5) were added to a testtube labelled “Enz Control”; and 50 μl of 10 mM Tris (pH 7.5) were addedto a test tube labelled “Blk.” Substrate solution (250 μl) containingcholesterol [¹⁴C]-oleate vesicles and sodium taurocholate in 150 mMTris, pH 7.5, were prepared as described and pipetted into each of thetest tubes described above. Cox, D., Leung, C. K. T., Kyger, E.,Spilburg, C., & Lange, L. (1990) Biochemistry 29, 3842. Human CEase,prepared as described in Example 3, was removed from the −80° C.freezer, thawed in an ice water bath and diluted with 400 μl of bufferconsisting of 1 part 150 mM Tris, pH 7.5, and 7 parts 100 mM sodiumtaurocholate in 150 mM Tris, pH 7.5. A 20 μl aliquot of CEase was thenadded to the test tubes, except the one labelled “Blk,” and the testtube rack was placed immediately in a 37° C. water bath. After tenminutes, the test tube rack was plunged into an ice water bath and theassay was completed as described elsewhere Cox, D., Leung. C. K. T.,Kyger, E. Spilburg, C. & Lange. L. (1990) Biochemistry 29, 3842.

To calculate the percent activity, the following formula was used:

$y = {{{Percent}\mspace{14mu}{Activity}} = \frac{{{CPM}\mspace{14mu}{Sample}} - {{{CPM}\;}^{``}{BLK}^{"}}}{{{{CPM}\;}^{``}{ENZ}\mspace{14mu}{Control}^{"}} - {{{CPM}\;}^{``}{BLK}^{"}}}}$

Using y as % activity and c as the concentration of very high molecularweight sulfated polysaccharide in the assay, the data were plottedaccording to the following function:log c=log (1/y−1)

The best straight line was drawn through the data points, and the IC₅₀was defined as the antilog of the x-intercept.

EXAMPLE 5

This example describes methods for characterizing the very highmolecular weight sulfated polysaccharides of this invention.

Determination of Degree of Substitution

Dowex-50W ion-exchange resin (H⁺ form, dry mesh 200–400; 8% crosslinkage) was added with gentle swirling to a 100 ml beaker containing 50ml of deionized water. The water was removed and the procedure wasrepeated two more times. The resin was added to a 1.0×20 cm column to abed height of 18 cm, and the column was washed with 25 ml of deionizedwater using a peristaltic pump at a flow rate of 30 ml/hr.

A 1.0 mg/ml solution (15 ml) of a very high molecular weight sulfatedpolysaccharide in water was pumped onto the resin and 5 minute fractionswere collected. When all of the sample was applied to the resin, the pHof each fraction was measured with a calibrated pH electrode. Thosefractions with a pH less than or equal to 3.5 contained protonatedsulfated polysaccharides and were pooled in a 50 ml glass beaker.

A rinsed conductivity electrode was immersed in the beaker containingthe protonated sulfated polysaccharide and the initial conductivityreading was recorded. The solution was titrated by recording theconductivity after each addition of 100 μl of 0.1 N NaOH. As base wasadded, the conductivity decreased until the equivalence point wasreached, then the conductivity increased. The equivalence point wasdetermined by drawing a straight line through the descending data pointsand a straight line through the ascending data points. The intersectionpoint of the two lines is the equivalence point, expressed as mls of0.10 N NaOH.

After completion of the titration, the amount of sulfated polysaccharidepresent was determined spectrophotometrically using Toluidine Blue. Indetail, 200 μl of sulfated polysaccharide solutions, ranging inconcentration from 2.5 μg/ml to 40 μg/ml, were pipetted into test tubes.A blank was prepared which contained only 200 μl of water, and variousaliquots were removed from the titration and the volume was adjusted to200 μl by adding an appropriate volume of water. After adding 10 μl of 1mg/ml Toluidine Blue to each tube, the absorbance was read at 540 nm,after zeroing against the blank. A standard curve was prepared and theamount of sulfated polysaccharide in a sample was determined from thelinear portion of the curve. Using this value and the equivalence point,the % sulfate can be determined from the following relation:

$\frac{{\%\mspace{14mu}{SO}_{3}} = \left( {8 \times {mls}\mspace{14mu}{NaOH}\mspace{14mu}{equivalence}\mspace{14mu}{{pt}.}} \right)}{\left( {{mg}\mspace{14mu}{{sulf}.\mspace{14mu}{polysaccharide}}\mspace{14mu}{toluidine}\mspace{14mu}{blue}\mspace{14mu}{assay}} \right)}$

The degree of substitution is defined as the number of hydroxyl groupson the polysaccharide that have been replaced by the OSO₃H functionalgroup. Every OH group which is lost is replaced by an OSO₃H group,increasing the molecular weight by 80. Since the molecular weight of astarting cellulose monomer is 161, the molecular weight (MW) increasesaccording to the following relation, where x=degree of the substitution:MW=161+80x

As sulfate is introduced into the polymer its percentage (y) changesaccording to the following relation:y=80x/(161+80x)

When this equation is solved for x, the degree of substitution can becalculated from the percent SO₃ in the sample.x=161y/80(1−y)Molecular Weight Determination

The molecular weight profile of a very high molecular weight sulfatedpolysaccharide is determined by aqueous gel permeation chromatographyusing a glucose-polydivinyl benzene GPC-HPLC column. Since the sulfatedpolysaccharide of this invention has a very high molecular weight andviscosity, the column is run at elevated temperatures to lower theviscosity to prevent pressure problems. Importantly, columns of thistype can be calibrated using standards of known molecular weight,allowing the molecular weight of an unknown sample to be determined bycomparing its elution volume to those of samples of known molecularweight. This HPLC assay is used to determine the molecular weight rangeof a high molecular weight sulfated polysaccharide and a cumulativeweight fraction plot is used to calculate the percentage low molecularweight compounds.

A mobile phase solution was prepared by adding 200 ml of DMSO to 800 mlof 0.1 M NaOH and then the solution was filtered through a 0.2 μmfilter. Molecular weight standard solutions were prepared by dissolvingindividual molecular weight standards in mobile phase solution to yielda concentration of 1 mg/ml. Finally, a sample solution of a very highmolecular weight sulfated polysaccharide was prepared by dissolving thesulfated polysaccharide in the mobile phase solution to yield aconcentration of 1 mg/ml. The samples were analyzed by injecting 500 μlof each individual standard in descending order of molecular weightvalue and then injecting 500 μl of the sample solution. The column wasoperated at 80° C.

A standard curve was prepared by plotting log₁₀ (M_(p)) of the standardswith known molecular weight versus their elution time. The equationdescribing the standard curve was calculated by the method of leastsquares. The log₁₀ (M_(p)) of the sulfated polysaccharide sample wasthen determined from its elution time and the derived equation.

The percentage of low molecular weight sulfated compounds is calculatedusing the following equation:% Low Molecular Weight=(AUC _(small) /AUC _(total))*100Where:

-   -   AUC_(total)=integration of the total area under the curve of the        sample peak.    -   AUC_(small)=integration of the area under the curve of the        sample peak from the elution time of the 75,000 Daltons standard        to the end of the curve.

EXAMPLE 6

Infra-red spectroscopy is used to verify the presence of sulfated groupsin the very high molecular weight sulfated polysaccharides prepared bythis invention. This example details the method to produce a Fouriertransform infrared (FTIR) spectrum of very high molecular weightsulfated polysaccharides prepared by the methods of this invention.

A sulfated polysaccharide/potassium bromide sample pellet was preparedby adding approximately 5 mg of solid sulfated polysaccharide and 495 mgof oven dried KBr into a polystyrene vial containing one plexiglassball. The solids were mixed with a Wig-L-Bug (International CrystalLaboratories), and 200 mg were loaded into a pellet die. A clear pelletwas prepared by subjecting the evacuated die to 6 metric tons ofpressure for 10 minutes. The clear pellet was removed from the die andplaced in the FITR sample chamber.

The sample spectrum, (FIG. 3), can be visually inspected to verify thepresence of certain characteristic absorptions. At about 800 cm⁻¹ thereis a distinct peak due to C—O—S stretching and at about 1240 cm⁻¹ thereis a distinct peak due to the S═O bond stretch. A reference spectrum ofcotton linter, (FIG. 3, bottom), shows the presence of these new bondsdue to the sulfate group.

EXAMPLE 7

This example demonstrates that very high molecular weight cellulosesulfate prepared by the method of this invention is an inhibitor ofcholesterol uptake into cultured human Caco-2 cells.

Colonic adenocarcinoma cells (Caco-2 cells; American Type CultureCollection) were grown to confluence (2.0×10⁶ cells per well) in plasticwells (22.6 mm; 4 cm²) and incubated overnight in Eagle's minimumessential medium and 10% lipoprotein deficient serum. The cells wererinsed once with 500 ml of PBS and then incubated with 8 mM sodiumtaurocholate, 1% bovine serum albumin and 1.0 pmole of [³H] cholesterolincorporated in phosphatidylcholine vesicles and various concentrationsof high molecular weight cellulose sulfate. The experiment was initiatedwith the addition of human cholesterol esterase to give a final enzymeconcentration of 200 nM in a reaction volume of 500 μl. At varioustimes, the reaction was quenched by removing the incubation medium andrinsing the cells with PBS. The cells were detached from the wells with1% sodium dodecyl sulfate solution (200 μl) and the cellular debriscounted to determine the amount of cholesterol associated with thecells. As shown in FIG. 4, incubation of homogeneous human pancreaticcholesterol esterase (200 nM) with [³H]-cholesterol in liposomes in thepresence of 2×10⁶ Caco-2 cells led to incorporation of free cholesterol,an effect entirely eliminated in the presence of 200 nM cellulosesulfate.

EXAMPLE 8

In order for the very high molecular weight sulfated polysaccharides ofthis invention to interact with cholesterol esterase, they must firstpass through the stomach where they can experience pH values less than2.0. Since cellulose based compounds are less stable at acid pH, thisinvestigation was carried out to demonstrate that degradation and lossin potency did not occur to a significant degree under simulated gastricconditions.

A 1.0 mg/ml solution of a very high molecular weight sulfatedpolysaccharide was prepared in a simulated gastric fluid (7 mlconcentrated HCl, 3800 units pepsin and 2 g NaCl in 1 L of water), and a1.5 μl aliquot was removed for analysis. The aliquot was immediatelyanalyzed for its ability to inhibit the cholesterol esterase catalyzedhydrolysis of cholesterol [¹⁴C]-oleate (Example 3) and its molecularweight was determined (Example 5). The remaining solution was placed ina 37° C. water bath, and time 0 was recorded as the test tube was placedin the bath. At 1 hr, 2 hr, and 25 hr, aliquots were removed andanalyzed for potency, molecular weight and the percent with a molecularweight less than 75,000 Daltons. As shown in Table III, there is nochange in IC₅₀ over a two hour incubation period and, moreover, there islittle change in molecular weight. While the starting molecular weightwas 5,000,000 Daltons, there is large error at these high values sothere is probably no significant difference between this value and thevalues seen at 1 hr and 2 hr, 3,900,000 Daltons and 3,600,000 Daltons,respectively. However, after 25 hr, there is evidence of degradationwith the molecular weight decreasing to 850,000 Daltons, which isaccompanied by a 3-fold increase in the IC₅₀ from 21 ng/ml to 68 ng/ml.

Another measure of degradation is the percentage of carbohydrate whichappears below an arbitrary molecular weight. In this case, 75,000Daltons was chosen since this is understood as the value above which noabsorption occurs. As shown in Table III, after 2 hr, only about 1% ofthe sample is degraded to a molecular weight below this value, and evenafter 25 hr, this value has increased to only 3.4%.

TABLE III STABILITY WITH PEPSIN AT pH 1.5 AND 37° C. TIME (hrs) IC₅₀(ng/ml) Molecular Wt. (kDa) % .75 kDa 0 26 5000 0.0 1 23 3900 0.4 2 213600 1.1 25 68 850 3.4

Taken together, this example indicates that over the residence timescommonly occurring in the stomach, the very high molecular weightsulfated polysaccharides of this invention do not lose their potency,and moreover, the sulfated polysaccharides are minimally degraded.

EXAMPLE 9

The objective of this study was to determine the amount of absorption oforally administered [¹⁴C]-labeled, very high molecular weight sulfatedpolysaccharides in male rats. The [¹⁴C]-labeled cellulose used in thisstudy was isolated from cotton bolls which had been exposed to ¹⁴CO₂,and the polysaccharide was sulfated following the procedure given inExample 1.

Six male Sprague-Dawley rats were given a single 375 mg/kg dose ofsulfated [¹⁴C]-labeled cellulose by oral gavage (Table IV).

TABLE IV DOSE SOLUTION ANALYSIS Parent Compound (mg/ml) 25.0Radioactivity (DPM/ml) 412898 Radioactivity (μCi/ml) 0.186 Activity(DPM/mg) 16516 Total Dose Administered (mg) 110Following dose administration, animals were placed in Elizabethancollars and fitted with fecal cups to prevent fecal contamination ofcollected urine. Cumulative urine samples were collected from 0–4, 4–8,and 8–24 hours post-dose. Feces were removed from the fecal cups at 12hours and 24 hours post-dose. Serial blood samples were obtained at0.33, 1, 3, 6, 10, and 24 hours following dose administration. Inaddition, a thorough cage-wash was performed following the last samplecollection. Derived plasma, urine, cage wash, feces and dose solutionwere assayed for radioactive content by oxidation followed byscintillation counting. The results were used to assess the oralabsorption of radioactivity following single oral dose administration ofhigh molecular weight sulfated [¹⁴C]-cellulose.

Radioactivity levels were not detectable in any of the plasma, urine andcage wash samples collected during the study. From the amount ofradioactivity administered and the detection limit of the method, inthis study, greater than 99.5% of the very high molecular weightsulfated polysaccharide was not absorbed.

EXAMPLE 10

This example demonstrates the importance of controlling the sulfationreaction temperature between 13° and 20° C.

Cotton linter cellulose was received from Buckeye Cellulose (Memphis,Tenn.) and DMF-SO₃ complex was from Du Pont (large scale reactor) orAldrich Chemical (bench scale).

The molecular weight of the cellulose sulfate polymer and the percentagewith a molecular weight less than 75,000 Daltons were determined by HPLCgel permeation chromatography as described in Example 5. The degree ofsulfation was determined using the conductometric titration described inExample 5.

Three samples (300 mg each) of minced cotton linters were soaked at 20°C. for 3 hour in 7.6 ml of anhydrous DMF. The flasks were immersed inwater baths at 15° C., 20° C. and 25° C. After standing for 30 min toreach temperature equilibrium, 1.14 g of DMF-SO₃ complex dissolved in2.5 ml DMF was added to each flask. After 3 hrs, the reactions werequenched by the addition of 915 mg of sodium bicarbonate followed by 25ml of water. The samples were stirred at ambient temperature for 20hours and then transferred to dialysis membranes (Molecular weight cutoff 10,000 Daltons). The samples were dialyzed exhaustively againstwater, lyophilized and the following properties were determined:molecular weight, % with molecular weight less than 75,000 Daltons,degree of sulfation, and elemental analysis. As summarized in Table Vbelow, a lower reaction temperature favors the formation of highmolecular weight polymer with less low molecular weight contamination.

TABLE V Properties of Cellulose Sulfate Synthesized at DifferentTemperatures Temp. Mol. Wt (kDa) % <75,000 Da SO₄/Mon. % Sulfur 15 9660.48 1.84 18.08 20 607 0.74 1.52 18.17 25 450 0.98 1.65 18.30

Sulfation of cotton linter cellulose was performed on a large scaleunder a blanket of nitrogen at a variety of temperatures following theprocedure described in Example 1. The maximum reaction temperature wasrecorded and the results are summarized in Table VI below.

TABLE VI Properties of Cellulose Sulfate Manufactured at VariousTemperatures Temp. Mol. Wt. SO₄ to Test No. Max (kDa) % <75 kDa MonomerYield (%) 1 16° 6160 <1 1.65 91 2 16° 1526 16.1 2.01 85 3 17° 3712 2.22.28 100 4 19° 3300 0.0 1.65 62 5 20° 1024 2.5 2.06 100 6 22° 929 4.01.94 74 7 25° 527 7.0 2.14 80 8 27° 394 8.54 2.04 100 9 27° 242 16.42.29 100 10 27° 324 11.1 1.95 92

The results indicate that the yield and degree of sulfation are bothinsensitive to temperature over the narrow range of 16° C. to 27° C. Theaverage degree of sulfation was 2.00, and under these reactionconditions, there was no trend indicating that temperature affects thisparameter. On the other hand, as evidenced by the decrease in molecularweight, cellulose sulfate underwent marked depolymerization over thissame narrow temperature range. Since low molecular weightpolysaccharides can be absorbed by the small intestine, the presence ofthese reaction by-products are of more serious concern than the averagemolecular weight, and, as shown in the table above, the higher reactiontemperature also favored the generation of these potentially toxicsubstances. Thus, when the maximum reaction temperature was 16° C.–19°C., only 1% to 2% of the sulfated material had a molecular weight lessthan 75,000 Daltons, while at 27° C. this value increased to 10%–15%.Taken together, these data indicate that when sulfation is carried outwith DMF-SO₃ complex, the temperature of the sulfation reaction shouldbe less than 20° C.

To define the minimum reaction temperature, the procedure described inExample 1 was followed except the reaction mixture was cooled to 1° C.(Step A) and the temperature was never allowed to exceed 13° C.throughout the 150 min reaction time. In every other way, themanufacturing run was identical to those described above. Following thisprocedure, the sulfated polysaccharide had a molecular weight of5,000,000 Daltons, but the yield was only 18.5%. Therefore, to producesulfated polysaccharide of high molecular weight and in good yield, thereaction temperature must be between 13° and 20° C.

EXAMPLE 11

Toxicity studies by the oral route have been carried out in rats anddogs with very high molecular weight sulfated polysaccharides of thisinvention. All studies reported here were conducted in compliance withthe Good Laboratory Practice Regulation set forth in 21 CFR 58. Twotypes of studies were performed. First, an acute study (dosed every 2hours for 24 hours) with a 14-day observation period was carried out inrats. Second, a chronic study was carried out in which high molecularweight sulfated polysaccharide was administered TID at daily dose levelsof up to 1,125 mg/kg in the rat and of up to 2,700 mg/kg in the dog.

Acute Administration (Rat). Ten male and ten female CD® rats wereassigned to either a control group or to a very high molecular weightsulfated polysaccharide treated group. Sulfated polysaccharide treatedanimals received by gavage 250 mg/kg every 2 hours throughout the courseof the day for a total dose of 3,250 mg/kg. Control animals received anequivalent volume of vehicle (deionized water) only. In this acutestudy, the high viscosity of the drug limited the dose solutionconcentration to 25 mg/ml. Given the dose volumes administered (10ml/kg) and the total number of doses received by each animal during thecourse of the day (13), the highest possible dose that could beadministered in one day was 3,250 mg/kg. The animals were observed for14 days and then subjected to necropsy. With the exception of transientsoft stools in three animals, there were no adverse findingsattributable to the drug. Parameters evaluated were mortality,morbidity, body weight, clinical signs and gross pathology. Theseresults are found in Table VII below.

TABLE VII Summary of Acute Oral Toxicity Study mg/kg/dose Group # ofCellulose ID # Animals Treatment Dosage Sulfate mg/kg/total Results 110M, DI water 10 ml/kg 0 0 No 10F Adverse Effects 2 10M, Cellulose 10ml/kg 250* 3250 No 10F Sulfate Adverse Effects** *Sulfatedpolysaccharides were administered every 2 hours over the course of 1day. **Three treated animals exhibited transient soft stools.

Chronic Administration (Rat). A very high molecular weight sulfatedpolysaccharide of this invention prepared by the method of Example 1 wasadministered orally by gavage to 2 groups of 15 male and 15 femaleCharles River CD® rats at dosage levels of 150 and 375 mg/kg three timesdaily for total dosage levels of 450 and 1,125 mg/kg/day. The controlgroup, consisting of 15 male and 15 female animals, received vehicle(deionized water) on a comparable regimen. Following 28 days oftreatment, 10 animals/sex/group were euthanized and fiveanimals/sex/group were allowed to recover for 14 days, and then theywere euthanized. Parameters evaluated were: mortality, clinical signs,body weight, food consumption, ophthalmoscopic examination, hematology,biochemistry, urinalysis, organ weights, and macroscopic and microscopicexamination of designated tissues. Statistical analysis was conducted onbody weight, food consumption, hematology, biochemistry, urinalysisparameters and organ weights. Criteria evaluated during the 14-dayrecovery period included all of the above except for ophthalmoscopicsigns.

Following four weeks of treatment and two weeks of recovery, bodyweight, food consumption and food efficiency values from all treatmentgroups were comparable to those of the control groups with nosignificant trends. The results are found in Table VIII below.

TABLE VIII Summary of 28-Day Oral Toxicity Study in CD Rats # of Dosagelevel Dose Dose Duration Animals (mg/kg/day)* Volume Solution (days)Results 15M, 15F 0 15 ml/kg DI water 28 NSE** 15M, 15F 450 15 ml/kg 10mg/ml 28 NSE 15M, 15F 1125 15 ml/kg 25 mg/ml 28 NSE *Each dose wasadministered in 3 equal portions each day. **NSE = No SignificantEffects

In summary, clinical pathology evaluation of all groups showed no testarticle-related findings in any of the treated groups. Anatomicpathology evaluation showed no test article-related organ weight changesand no test article-related microscopic observations in any organs ortissues examined.

Chronic Administration (Dogs). Very high molecular weight sulfatedpolysaccharides prepared following the method of Example 1 wereadministered orally for 28 days via gelatin capsules to groups of 3 to 4purebred beagle dogs/sex at dosage levels of 100, 300 and 900 mg/kg TIDfor total dosage levels of 300, 900 and 2,700 mg/kg/day. The controlgroup received empty gelatin capsules. Following 28 days of treatment, 3dogs/sex/group were necropsied. The remaining 1 dog/sex in the control,900 and 2,700 mg/kg/day groups were held for a 17-day recovery periodand then euthanized.

Detailed clinical examinations were made once a week. All animals wereobserved for mortality, morbidity, and overt signs of toxicity twice aday and for pharmacotoxic signs just prior to dosing and about 2 hourspost dose. Body weights and food consumption were recorded pretest andweekly. Complete physical examinations were conducted during pretest andat the end of the dosing and recovery phase. Ophthalmoscopic andelectrocardiographic examinations were conducted during theacclimatization period and at the end of the dosing phase. Clinicalpathology laboratory studies (hematology, serum biochemistry andurinalysis) were conducted once during pretest and at the end of thedosing and recovery periods. Complete macroscopic pathologicexaminations were performed on all animals at the scheduled necropsiesfollowing the dosing and recovery periods. Absolute and relative organweights were recorded for selected organs. Microscopic examinations wereperformed on selected tissues for all control and high dose animals.

All of the animals survived to study termination. Test article-relatedclinical signs included transient emesis in one male and soft stool andunformed feces of liquid consistency. The incidence of emesis wasincreased in males at the 2,700 mg/kg/day dosage level in comparison tothe controls. A dosage-related increase in soft stool was noted, mainlyat the 900 and 2,700 mg/kg/day dosage levels. Male and female dogsreceiving 2,700 mg/kg/day had markedly increased incidence of unformedliquid stools relative to controls; the incidences observed in the 300and 900 mg/kg/day dosage level groups were marginally increased comparedto controls. In spite of these findings, no meaningful differences wereobserved in body weights or food consumption during the 4-week dosingperiod. During the recovery period, the incidence of these clinicalsigns were similar in all groups. The results of the testing are shownin Table IX below.

TABLE IX # of Dosage Level Dose Capsule Duration Animals (mg/kg/day)*Volume Volume (days) Results 4M, 4F 0 4 capsules empty 28 NAE** 3M, 3F300 4 capsules BPC*** 28 NAE 4M, 4F 900 4 capsules BPC 28 loose stools4M, 4F 2700 4 capsules BPC 28 loose stools**** *Each dose wasadministered in 3 equal portions each day. **NAE = No Adverse Effects.***High molecular weight sulfated polysaccharide. ****Transient emesiswas noted in one male.No toxicologically significant or test article-related findings werenoted in the following: physical, ophthalmoscopic andelectrocardiographic examinations; hematological, biochemical andurological parameters; organ weights; macroscopic and microscopicpathology. Thus, no evidence of systemic toxicity was detected in maleand female dogs after 28 days of oral dosing of high molecular weightsulfated polysaccharides via capsule at levels up to 900 mg/kg TID(2,700 mg/kg/day).

EXAMPLE 12

A very high molecular weight sulfated polysaccharide of this invention,prepared by the method of Example 1, was tested for mutagenic activityin the Salmonella-Escherichia coli/mammalian-microsome reverse mutationassay, in the L5178Y TK+/−mouse lymphoma forward mutation assay and inan in vivo mouse micronuclease assay.

Salmonella-Escherichia coli/Mammalian-Microsome Reverse Mutation Assay(Ames Test). This assay evaluates the high molecular weight sulfatedpolysaccharide and/or its metabolites for their ability to inducereverse mutations in the genome of specific Salmonella typhimuriumtester strains and an Escherichia coli tester strain, both in thepresence and absence of an exogenenous metabolic activation system ofmammalian microsomal enzymes derived from Aroclor™ induced rat liver(S9). The tester strains used in the mutagenicity study were Salmonellatyphimurium TA98, TA100, TA1535, TA1537, TA1538 and Escherichia colitester strain WP2uvrA⁻. Each assay was conducted using six doses of highmolecular weight sulfated polysaccharide, three plates per dose, alongwith a concurrent vehicle (deionized water) and positive and negativecontrols in both the presence and absence of S9 mix. The doses of testartice tested in this study were 66.7, 100, 333, 667, 1,000 and 1,500 μgper plate. The experimental findings are shown in Table X below.

TABLE X Summary of Results of the Ames Test Organisms HSP* (μg/plate) S9Results S. typh. TA 98 67–1,500 + − TA 98 67–1,500 − − TA 100 67–1,500 +− TA 100 67–1,500 − − TA 1535 67–1,500 + − TA 1535 67–1,500 − − TA 153767–1,500 + − TA 1537 67–1,500 − − TA 1538 67–1,500 + − TA 1538 67–1,500− − E. coli WP2uviA 67–1,500 + − WP2uviA 67–1,500 − − *HSP Highmolecular weight sulfated polysaccharide

The results in Table X indicate that under the conditions of this studyhigh molecular weight sulfated polysaccharides do not cause a positiveincrease in the number of revertants per plate of any of the testerstrains either in the presence or absence of microsomal enzymes preparedfrom rat liver (S9).

Mouse Lymphoma Forward Mutation Assay. This in vitro assay evaluates theability of test articles to induce forward mutations at the thymidinekinase (TK) locus in the mouse lymphoma L5178Y cell line. A singlemutation assay was performed for both nonactivation and rat liver S9metabolic activation conditions. Six treatments from 500 μg/ml to 5000μg/ml were initiated with and without activation. At most, weakcytotoxicities were induced. Under nonactivation and activationconditions, none of the six assayed treatments induced a mutantfrequency that exceeded the minimum criterion for a positive responseand no dose-related trend was observed. Therefore, high molecular weightsulfated polysaccharides are considered to be negative for inducingforward mutations at the TK locus in L5178Y mouse lymphoma cells underthe nonactivation and S9 metabolic activation conditions used in thisstudy.

In Vivo Mouse Micronuclease Assay. This assay evaluates the ability oftest articles to induce micronuclei in bone marrow polychromaticerythrocytes of CD-1 (ICR) mice. For the assay, high molecular weightsulfated polysaccharide dose levels of 800, 1600 and 3200 mg/kg wereselected. Ten animals (five males and five females) were randomlyassigned to each dose/harvest time group and dosed at 40 ml/kg. Positivecontrol groups were euthanized approximately 24 hours after dosing. Theanimals dosed with the high molecular weight sulfated polysaccharidewere euthanized at 24, 48 and 72 hours after dosing for extraction ofthe bone marrow. The experimental findings are shown in Table XI below.

TABLE XII Micronucleas Test Data Summary % Micronucleated PCEs** HarvestTime Mean of 1000 per animal ± S.E. Treatment Dose (HR) Males FemalesTotal Vehicle Control 40 mg/kg 24 0.00 ± 0.00 0.02 ± 0.02 0.01 ± 0.01Sterile Deionized 48 0.04 ± 0.04 0.06 ± 0.04 0.05 ± 0.03 Water 72 0.04 ±0.02 0.04 ± 0.02 0.04 ± 0.02 Positive Control 80 mg/kg 24  2.20 ± 0.46* 2.22 ± 0.25*  2.21 ± 0.25* Cyclophosphamide HSP*** 800 mg/kg 24 0.00 ±0.00 0.08 ± 0.06 0.04 ± 0.03 48 0.08 ± 0.06 0.06 ± 0.04 0.07 ± 0.03 720.02 ± 0.02 0.12 ± 0.06 0.07 ± 0.03 1600 mg/kg 24 0.10 ± 0.06 0.08 ±0.04 0.09 ± 0.03 48 0.02 ± 0.02 0.00 ± 0.00 0.01 ± 0.01 72 0.02 ± 0.020.02 ± 0.02 0.02 ± 0.01 3100 mg/kg 24 0.02 ± 0.02 0.00 ± 0.00 0.01 ±0.01 48 0.06 ± 0.04 0.02 ± 0.02 0.04 ± 0.02 72 0.04 ± 0.04 0.00 ± 0.000.02 ± 0.02 **PCE Polychromatic Erythrocyte. ***HSP High MolecularWeight Sulfated Polysaccharide.From these data, it is concluded that the high molecular weight sulfatedpolysaccharide used here does not induce a significant increase inmicronuclei in bone marrow polychromatic erythrocytes under theconditions of this assay, and it is considered negative in the mousebone marrow micronucleus test.

EXAMPLE 13

We have found that administering a very high molecular weight sulfatedpolysaccharide as prepared in Example 1 to humans in an amount of about1000 mg at or about meal time lowers both total cholesterol and LDL.

Five human subjects comprising males or females between the ages of21–70 were selected for the study population. Excluded from thepopulation were persons having a history of medical disease and drugabuse, females with child bearing potential, any subject who had taken adose of any medication within two weeks of the study, any person with abody weight more than 30% above or 20% below Metropolitan Life InsuranceCo. Tables, any subject who uses or used tobacco products in the pastyear, and any person who is a subject in another therapeutic agent trialor who has been in the last 30 days.

An essentially non-absorbable very high molecular weight sulfatedpolysaccharide as prepared in Example 1 was supplied in powdered formand 1000 mg were added to 8 ounces of a prepared commercial diet softdrink (such as CRYSTAL LIGHT®) that was previously mixed in boilingwater. The powdered sulfated polysaccharide was stirred into the liquidmixture for up to twenty minutes or until it went into solution.Finally, the solution was allowed to cool before administration to thehuman subject.

The prepared dose was administered three times per day just prior to ameal at 8:00 AM, 12 noon, and 6:00 PM. This exact dosing schedule wasfollowed for each of the 7 days of the trial.

Serum samples were taken from each subject immediately before the firstdose, at day 1, day 4, day 8, and day 14 and each sample was analyzedfor total cholesterol and LDL. The results are found in Table XII below.

TABLE XII 1000 mg Dosage Results* Baseline Day 4 Day 8 Day 14 SubjectTotal Total Total Total No. Chol. LDL Chol. LDL Chol. LDL Chol. LDL 1185 128 170 122 183 128 169 115 2 252 194 229 176 226 148 230 156 3 253173 254 205 247 186 234 151 4 209 152 192 141 197 140 186 129 5 188 132164 121 168 117 149 95 Σ 1087 779 1009 765 1021 719 968 646 mean 217 156202 153 204 144 194 129 std. dev. 33 28 39 37 32 26 37 25 *mg/dl.

The same analyses were performed on a group of subjects taking a placebo(only CRYSTAL LIGHT®) in the same manner as that described above. Theseplacebo results are found in Table XIII below.

TABLE XIII Placebo Dosage Results* Baseline Day 4 Day 8 Day 14 SubjectTotal Total Total Total No. Chol. LDL Chol. LDL Chol. LDL Chol. LDL 302223 153 219 134 197 143 205 141 305 154 97 155 99 152 106 166 108 307149 100 142 90 133 86 166 108 310 296 219 275 183 259 183 275 201 313244 175 245 166 230 158 222 141 316 272 190 257 167 265 175 261 190 317251 157 259 176 296 203 276 194 321 228 154 228 155 241 153 202 125 233199 146 190 133 194 122 202 139 326 177 114 174 100 155 82 162 100 329251 167 235 145 220 141 236 163 331 240 175 224 163 213 135 235 145 Σ2684 1647 2603 1711 2555 1678 2608 1755 mean 224 154 217 143 213 140 217146 std. dev. 46 36 43 32 49 37 41 35 *mg/dl.The cholesterol levels from the placebo experiment and from the highmolecular weight sulfated polysaccharide are summarized in Table XIVbelow.

TABLE XIV Comparison of Placebo and HSP* on Serum and LDL- CholesterolLevels** Total Cholesterol LDL-Cholesterol Time Placebo HSP* PlaceboHSP* Baseline 224 217 154 156 Day 4 217 202 143 153 Day 8 213 204 140144 Day 14 217 194 146 129 *HSP High molecular weight sulfatedpolysaccharide **mg/dlThe data indicate that the high molecular weight sulfated polysaccharidemanufactured according to Example 1 lowers serum cholesterol 10.6% from217 mg/dl to 194 mg/dl and it also lowers LDL-cholesterol 17.3% from 156mg/dl to 129 mg/dl.

1. A method for lowering serum cholesterol in humans comprisingadministering to a human the combination of an essentiallynon-absorbable very high molecular weight sulfated polysaccharide havingless than about 0.98 wt. percent of sulfated polysaccharides having amolecular weight less than 75,000 Daltons and containing less than 0.5weight percent of inorganic sulfate and a second compound that reducesserum cholesterol levels wherein the high molecular weight sulfatedpolysaccharide is prepared by sulfating a very high molecular weightpolysaccharide with a sulfur containing compound at a temperate lessthan about 20° C.
 2. The method of claim 1 wherein the second compoundis at least one cholesterol synthesis blocker.
 3. The method of claim 2wherein the cholesterol synthesis blocker is lovastatin.
 4. The methodof claim 1 wherein the second compound is an inhibitor of ACAT.
 5. Themethod of claim 1 wherein the sulfated polysaccharide is sulfatedcellulose.
 6. A method for lowering serum cholesterol in humanscomprising administering to a human the combination of an essentiallynon-absorbable very high molecular weight sulfated cellulose having lessthan about 0.98 wt. percent of sulfated cellulose having a molecularweight less than 75,000 Daltons and containing less than 0.5 weightpercent of inorganic sulfate and lovastatin wherein the high molecularweight sulfated polysaccharide is prepared by sulfating a very highmolecular weight polysaccharide with a sulfur containing compound at atemperature less than about 20° C.